The present invention relates mediums for the growth of microorganisms which are effective for improving plant productivity. More particularly, the invention provides a legume medium effective for the cultivation of agronomically beneficial microbes, such as Azospirillum.
A number of microorganisms are known to have beneficial effects on plant growth. Among these are nitrogen fixing bacteria of the Rhizobium species, which are symbionts of leguminous species. Azospirillum species, which are free living nitrogen fixing bacteria associated with the roots of grasses, are also now recognized for their plant growth promoting qualities (Mishustin and Shilnikova, Moscow, Science Publ. House, 1973; Mishustin and Shilnikova, Moscow, Science Publ. House, 1968). More specifically, certain strains of Azospirillum brasilense have been shown to enhance accumulation of various minerals in wheat and soybean (Bashan et al., Applied and Environ. Microbiol., 56(3):769-775 (1990)), increase dry weights of maize shoots (O""Hara et al., Can. J. Microbiol., 27:871-877 (1981), and increase dry weights of sorghum, pearl millet and napier grass (Smith et al., Applied and Environ. Microbiol., 47(6):1331-1336 (1984)).
Inoculation of seeds or soil with beneficial microorganisms, including Azospirillum, for crop improvement has been practiced for a number of years. However, variable and inconsistent results have often been observed possibly due to loss of inoculant viability or variability of dosage due to changes in inoculant viability (Okon et al., CRC Crit. Rev. Biotechnology, 6:61-85 (1987)).
Successful inoculation experiments appear to be those in which researchers have paid special attention to the optimal number of cells in the inoculant, using appropriate procedures to assure that cells remain viable for colonizing the roots (ASM News, Vol. 63, No. 7). Hence, the manner in which agronomically beneficial microorganisms are grown is critical to their viability and ultimate success in improving plant productivity.
Agronomically beneficial microbes have traditionally been cultured in synthetic mediums. For example, one of the most common mediums used for growing Azospirillum is NFb medium or a modification of NFb (Okon et al., Soil. Biol. Biochem., vol. 26, No. 12, pp. 1591-1601, 1994). These types of mediums do not provide microbes that consistently improve crop productivity. Further, synthetic mediums add to the cost of microbial inoculants.
The present invention is directed to a legume medium that is effective for optimal growth of microorganisms that are useful for increasing plant productivity and soil quality. Growth of agromonically benefical microbes on the legume medium of the present invention consistently and quickly provides high numbers of microbes in a physiological state that is optimal for increasing plant productivity. In an important aspect, the legume medium of the invention is effective for laboratory scale and large scale growth and propagation of microbes.
In accordance with the present invention, the legume medium is an aqueous extract of legumes that is prepared by boiling legume seeds, stems, leaves, and mixtures thereof in water. In this aspect of the invention, the legumes may include peas, beans, clover, alfalfa and mixtures thereof. In an important aspect of the invention, about 150 grams to about 250 grams of legumes are boiled in about one liter of water for about 15 to 30 minutes. After boiling, the medium is allowed to cool and solids are removed. The medium may be used directly for the propagation of agronomically beneficial microbes or optionally, may be further supplemented with a nitrogen source, a carbon source, or mixtures thereof.
In another important aspect, the present invention provides a method for growing agronomically beneficial microbes. In accordance with the method of the invention, an agronomically beneficial microbe is inoculated into a legume extract medium and cells are growth under conditions effective for providing a cell density of at least about 108 cells/ml.